I am often asked why reversed-phase TLC data does not translate well to reversed-phase flash column chromatography. There are several reasons for this and in this post I will attempt to explain the challenges associated with reverse-phase TLC as a method development tool for reversed-phase flash chromatography.
Reversed-phase flash chromatography is an increasingly utilized purification tool in medicinal and organic chemistry labs. However, using a reversed-phase TLC plate to help in method development is not always productive as the solute/stationary phase interactions are different than with reversed-phase column chromatography. "How so?" you may ask.
Well, there are some fundamental differences, normal-phase separations are based on an adsorption - desorption mechanism while reversed-phase separations operate using partitioning between the stationary and mobile phases to retain and selectively elute compounds.
What this means is that compounds, under normal-phase conditions, will adsorb onto the surface of the silica stationary phase. The mobile phase then solvates the compound off the silica during the elution gradient and the compounds do not re-adsorb onto the silica.
With reversed-phase, compounds continually interact (partition) between the mobile phase and the stationary phase, e.g. C18, but only if the waxy stationary phase has been "wetted" first with a suitable organic solvent (MeOH, MeCN). A poorly wetted C18 has reduced surface area which directly impacts the chromatography. This is the main problem with reversed-phase TLC. Let me explain a bit more...
- TLC plate surfaces must be dry in order for capillary action to pull the solvent and compounds up the surface. This is just fine when the primary mechanism for separation is adsorption as in normal-phase TLC. However, there is a significant difference between the way dry and wetted reversed-phase media interact with compounds. Because the elution solvents in reversed-phase are polar and the stationary phase a non-polar wax, there is very little ability for the solvent to permeate the reversed-phase layer. This lack of surface area and permeability results in restricted compound partitioning meaning solutes will tend to migrate with the solvent front or close to it. Therefore, the partitioning dynamics compared to an HPLC column are not consistent and methods cannot easily be transferred from reversed-phase TLC to reversed-phase flash.
- Since the reversed-phase TLC plate's waxy surface is hydrophobic, sample application can be a challenge unless a good wetting solvent is used (DCM, acetone, DMSO, DMF) or if the plate has a pre-adsorbing layer of diatomaceous earth. Unless the plate has the pre-adsorbent layer, water-dissolved samples cannot be applied to the reversed-phase plate.
What has been your experience with reversed-phase TLC as a method development tool? Have you had success? If so, please share your techniques.
For more information on flash chromatography, please download our white paper Successful Flash Chromatography.